Aptamer Selection Based on Microscale Electrophoretic Filtration Using a Hydrogel-Plugged Capillary Device.

This study reports a novel aptamer selection method based on microscale electrophoretic filtration. Aptamers are versatile materials that recognize specific targets and are attractive for their applications in biosensors, diagnosis, and therapy. However, their practical applications remain scarce due to issues with conventional selection methods, such as complicated operations, low-efficiency separation, and expensive apparatus. To overcome these drawbacks, a selection method based on microscale electrophoretic filtration using a capillary partially filled with hydrogel was developed. The electrophoretic filtration of model target proteins (immunoglobulin E (IgE)) using hydrogel, the electrokinetic injection of DNAs to interact with the trapped proteins, the elimination of DNAs with weak interactions, and the selective acquisition of aptamer candidates with strong interactions were successfully demonstrated, revealing the validity of the proposed concept. Two aptamer candidates for IgE were obtained after three selection cycles, and their affinity for the target was confirmed to be less than 1 nM based on their dissociation constant (KD) values. Therefore, the proposed method allows for the selection of aptamers with simple operations, highly effective separation based on electrophoresis and filtration, and a relatively cheap apparatus with disposable devices.

Direct Measurement of Initial Rate of Enzyme Reaction by Electrokinetic Filtration Using a Hydrogel-plugged Capillary Device.

A novel electrokinetic filtration device using a plugged hydrogel was developed to directly measure the initial rate of enzyme reactions. In the proposed method, the enzyme reaction proceeded only for a short time when the substrate was passed through a thin layer of enzyme trapped by the hydrogel without any lag times for mixing and detection. In experimental conditions, alkaline phosphatase (enzyme) was filtrated at a cathodic-side interface of the plugged hydrogel by molecular sieving effect, providing the thin enzyme zone whose thickness was approximately 100 µm. When 4-methylumberiferyl phosphate (substrate) was electrokinetically introduced into the device after trapping the enzyme, 4-methylumberiferone (product) was generated by the enzyme reaction for only 1.26 s as the substrate passed through the trapped enzyme zone. As a result, the initial rate of the enzyme reaction could be directly calculated to 31.0 µM/s by simply dividing the concentration of the product by the tunable reaction time. Compared to the initial rate obtained by mixing the enzyme and substrate solutions, the value of the maximum velocity of the enzyme reaction was 30-fold larger than that in the mixing method due to the preconcentration of the enzyme by trapping. The Michaelis-Menten constant in the proposed method was 2.7-fold larger than that in the mixing method, suggesting the variation of changes in the equilibrium of complex formation under the experimental conditions.